Introduction

Uveal melanoma is the most common intraocular malignant tumor in adults and comprises approximately 5% of all melanomas (1). Even after treatment of the primary tumor, 20% to 50% of patients succumb to metastatic disease. The liver is the predominant organ of metastasis. Standard chemotherapies and immune checkpoint blockers rarely induce clinical responses in patients with macrometastasis and their 1-year survival rate is less than 30% (2). This knowledge emphasizes an urgent unmet need for effective therapeutic strategies for advanced uveal melanoma.

A major effector pathway downstream of mutant Gαq and Gα11 is RAF–MEK1/2–ERK1/2 signaling. Inhibition of MEK1/2 with trametinib or selumetinib induces either cell-cycle arrest or apoptosis in uveal melanoma cell lines (7, 20); however, clinical studies in advanced-stage patients with uveal melanoma indicate that MEK inhibitors have limited clinical benefit. Trametinib was ineffective in a phase I trial cohort of 16 metastatic patients with uveal melanoma (21). A phase II trial with selumetinib in 120 patients showed a 9-week improvement in progression-free survival compared with standard chemotherapy but no improvement in overall survival (22). In the most recent phase III trial with 129 patients (23), a combination of selumetinib and standard chemotherapy dacarbazine failed to improve progression-free survival compared with chemotherapy alone. Thus, while MEK inhibitor may form part of a therapeutic approach for advanced-stage uveal melanoma, further investigation is required to identify inhibitors to act in combination.

The majority of patients with uveal melanoma with overt metastasis show primary resistance to MEK inhibitors, which may be mediated by factors from the tumor microenvironment. In uveal melanoma cell monocultures, hepatocyte growth factor (HGF) provides resistance to MEK inhibitors (20). HGF is secreted by quiescent hepatic stellate cells. Consistent with the presence of HGF in tumor microenvironment, the majority of uveal melanoma liver metastases express phosphorylated/activated cMET (20). Together, these results suggest that MEK inhibitors in combination with cMET targeting agents may have utility in advanced uveal melanoma. In this study, we explored the molecular mechanism of HGF-mediated resistance to MEK inhibitors in uveal melanoma cells and preclinically evaluated the efficacy of cotargeting cMET with MEK inhibitor in metastatic cell lines and ex vivo explants. Our data show that downregulation in the BH3-only proteins, Bim-EL and Bmf, contribute to HGF-mediated protective effect in metastatic uveal melanoma cells. Clinical grade cMET targeting agents effectively overcome the resistance provided by exogenous HGF as well as factors derived from hepatic stellate cells. Combined inhibition of cMET and MEK1/2 enhances apoptotic signal in cell lines and an ex vivo explant model of metastatic uveal melanoma. Together, these data provide a preclinical basis for combinational therapies targeting mutant Gαq/11 signaling and signaling initiated by factors from tumor microenvironment in advanced-stage patients with uveal melanoma.

Cell line validation

UM001 and UM004 cells were confirmed as harboring GNAQ mutations as determined by Sanger DNA sequencing. UM001 and UM004 cells were analyzed by STR analysis on January 15, 2015. The UM001 and UM004 profiles were unique, although the latter had a 94% match with 3 changed alleles to MDA-MB-330 cells on the DSMZ resource.

siRNA and transfection

UM004 cells (3 × 105) were seeded in 6-well plates overnight before transfection with chemically synthesized siRNAs at a final concentration of 25 nmol/L using Lipofectamine RNAiMAX (Invitrogen) as previously described (28). Bim-EL–specific siRNAs (GACCGAGAAGGUAGACAAUUGTT and CAAUUGUCUACCUUCUCGGUCTT) were purchased from Cell Signaling Technology. Bmf-specific siRNA (GAGUAACAGAUAACGAUUA) was purchased from Dharmacon Inc. A nontargeting siRNA (UAGCGACUAAACACAUCAAUU) was used as a control.

EdU incorporation assay

UM001 and UM004 cells were treated with DMSO, 50 nmol/L trametinib, 10 ng/mL HGF or trametinib plus HGF for 32 hours before the addition of 10 μmol/L EdU for another 16 hours. Cells were then processed using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen) according to the manufacturer's protocol. Experiments were performed in triplicate, and statistical analysis was performed using a 2-tailed t test assuming unequal variance with error bars representing SD.

Annexin V/propidium iodide staining

Cells were trypsinized, washed with cold PBS, and resuspended in 0.1-mL binding buffer. Cells were then stained with 5 μL of Annexin V-APC and 2 μL of propidium iodide (PI) for 30 minutes at room temperature. Staining was then analyzed by flow cytometry on a BD FACSCalibur flow cytometer (BD Biosciences). Data were analyzed by FlowJo software (Tree Star, Inc.). Experiments were performed in triplicate with statistical analysis as in EdU incorporation assay.

Crystal violet staining

Exponentially growing UM001 and UM004 cells were plated in 6-well or 12-well dishes and treated as described in figure legends. Cells were then stained with crystal violet solution (1% crystal violet, 10% buffered formalin) for 30 minutes, washed, and air dried. Plates were imaged by scanning while pictures were taken at 100× magnification on the Nikon Eclipse Ti inverted microscope with NIS-Elements AR 3.00 software (Nikon). Crystal violet staining images were quantitated using ImageJ. Experiments were performed in triplicate, and statistical analysis was performed using a 2-tailed t test assuming unequal variance

ELISA

HGF levels in conditioned medium collected from stellate cell cultures were measured with ELISA kits (Invitrogen), according to the manufacturer's instructions.

Migration and invasion assays

Subconfluent UM001 and UM004 cells were cultured overnight in serum-free medium. For migration assays, 1 × 104 cells in serum-free medium were placed inside 8.0-μm pore size cell culture inserts (BD Biosciences). For invasion assays, the inserts were first coated with 0.75 mg/mL Matrigel (BD Biosciences) for 1 hour before plating cells inside each chamber. Cells were allowed to migrate or invade for 16 hours toward an attractant of stellate cell culture medium or conditioned medium from passage 3 stellate cells. Chamber filters were fixed in buffered formalin and stained with crystal violet. Cells in the inner chamber were removed. Images were taken with a Nikon Eclipse Ti inverted microscope at 100× magnification.

Reverse-phase protein array analysis

UM001 and UM004 cells were plated in 6-well dishes at 4 × 105 cells per well. Cells were treated with unconditioned medium or passage 2 HHSteC conditioned medium for either 1 or 48 hours. Cells were lysed and prepared as previously described (29) and analyzed at the MD Anderson Functional Proteomic core facility (Houston, TX). Serial dilutions of samples were arrayed on nitrocellulose-coated slides and run against 295 validated antibodies. Spot density was determined using MiroVigene, and analyses of triplicate normalized data were performed using SuperCurve. Hierarchical clustering of the 295 antibodies was performed via the clustergram function in MATLAB (version 2015b) on reverse-phase protein array (RPPA) median–centered log2 expression values. The samples were presorted on the basis of cell line, treatment type, time point, and replicate number.

Ex vivo uveal melanoma explants

Human metastatic uveal melanoma tissue was collected following patient consent at Thomas Jefferson University Hospital (Philadelphia, PA) under an IRB-approved protocol (#02.9014R). Less than 16 hours postsurgery, excess adipose and stromal tissue were removed, and the tumor (Explant of Patient No.4/Ex-Pt#4) was cut into 1-mm3 pieces. Vetspon-absorbable hemostatic gelatin 1-cm3 sponges (Novartis) were presoaked in 12-well plates for 15 minutes at 37°C in 500 μL of DMEM medium containing 10% FBS, penicillin/streptomycin, and drugs. DMSO was used as a vehicle control. To avoid concerns of intratumoral heterogeneity, up to four ∼1-mm3 pieces from different locations of the original tumor were placed per sponge per treatment condition. Samples were treated for 48 hours with medium being replaced after 24 hours. Tumor pieces for Western blotting were homogenized in lysis buffer with phosphatase and protease inhibitors (PhosSTOP and cOmplete tablets, Roche). Laemmli sample buffer was added and samples were heated at 99°C for 5 minutes.

Statistical analysis

Statistical analyses were performed using SPSS v22.0 (IBM). One-way ANOVA analyses were performed on normalized data from groups of equal sizes. No outliers were identified during inspection of boxplots. All of the groups were determined to be normally distributed using Shapiro–Wilk test (P > 0.05). There was homogeneity of variances among all groups, as determined by Levene test of equality of variances (P > 0.05). Dunnett one-tailed multiple comparison post-hoc tests were performed to determine statistical significance.

We next analyzed the cell-cycle profiles of UM001 and UM004 cells treated with HGF, trametinib, or the combination of HGF plus trametinib. Trametinib treatment was associated with changes in G1–S regulators including lower expression of cyclin A2 and cyclin D1 and reduced retinoblastoma (RB) phosphorylation (Fig. 1C). Downregulation of total RB expression following trametinib treatment was also detected, an effect previously observed in breast cancer cell lines following inhibition of cell-cycle progression with CDK4/6 inhibitors (30, 31). MEK inhibition increased expression of the apoptotic markers, cleaved PARP and cleaved caspase-3. Notably, trametinib-treated cells treated with HGF showed a partial recovery of cyclin A2, cyclin D1, and phospho-RB levels. HGF also modestly increased levels of phosho-ERK1/2 in trametinib-treated cells. In addition, the induction of cleaved PARP and cleaved caspase-3 was mitigated by HGF in trametinib-treated cells (Fig. 1C). Together, these data indicate that HGF promotes the growth of trametinib-treated cells through restoration of cell-cycle progression and inhibition of apoptosis.

Downregulation of Bim-EL and Bmf contributes to HGF-mediated resistance to trametinib in uveal melanoma cells

To molecularly understand how HGF counteracts trametinib-mediated apoptosis, we compared the levels of Bcl-2 family proteins in uveal melanoma cells treated with HGF, trametinib, or the combination of both. We also treated cells with MK2206 to evaluate the role of AKT activity. HGF promoted the phosphorylation of AKT in the presence of trametinib, an effect that was diminished by MK2206 (Fig. 2A). Trametinib treatment did not alter expression of antiapoptotic proteins, Bcl-w and Bcl-xl, multidomain proapoptotic proteins, Bak and Bax, or BH3-only proteins, Bad, Bid, and Noxa, in UM001 and UM004 cells (Fig. 2A). In contrast, the BH3-only proapoptotic proteins, Bcl-2–interacting mediator of cell death extra large (Bim-EL), and Bcl-2 modifying factors (Bmf) were upregulated in trametinib-treated cells. The induction of Bim-EL and Bmf was diminished or markedly reduced when HGF was supplemented to trametinib-treated uveal melanoma cells (Fig. 2A). Notably, Bim-EL and Bmf levels were reinduced in cells treated with a combination of trametinib, HGF, and MK2206, suggesting that HGF activation of AKT mediates the resistance to trametinib. A modest upregulation of the prosurvival protein Bcl-2 and the BH3-only proapoptotic protein, Puma, was detected with trametinib treatment in one (Puma) or both (Bcl-2) cell lines (Fig. 2A).

To determine whether upregulation of Bim-EL and Bmf is required for trametinib-induced inhibition of cell viability, Bim-EL and/or Bmf-silenced UM004 cells were treated with trametinib (Fig. 2B) and evaluated by crystal violet staining (Fig. 2C). In comparison with controls, trametinib decreased cell viability by about 60% (Fig. 2C). Individual knockdown of Bim-EL and Bmf each partially rescued cells from trametinib with cell viability inhibited by 32% to 39%; however, simultaneous silencing of Bim-EL and Bmf further restored the viability of trametinib-treated cells with cell growth decreased by about 26% of the control (Fig. 2C). To examine whether Bim-EL and Bmf are sufficient to promote uveal melanoma cell apoptosis, UM001 and UM004 cells were infected with adenoviruses to express Bim-EL, Bmf, and enhanced green fluorescence protein (eGFP), as a control. Ectopic expression of Bim-EL and/or Bmf significantly increased apoptosis in uveal melanoma cells, whereas expression of eGFP showed little effect (Supplementary Fig. S2). These results suggest that Bim-EL and Bmf are sufficient to induce apoptosis and are downregulated in HGF-mediated resistance to MEK inhibitors.

To inhibit HGF-mediated signaling, we utilized 2 cMET targeting agents that are being tested in clinical trials for patients with uveal melanoma with liver metastasis as well as other advanced cancers. Of these 2 agents, LY2801653 is a type II kinase inhibitor with cMET as one of its target and displays antitumor activity in non–small cell lung carcinoma and cholangiocarcinoma preclinical models (32–34). LY2875358 is a neutralizing and internalizing anti-cMET bivalent antibody that showed potent antitumor activity in both HGF-dependent and cMET-amplified preclinical tumor models (35). Initially, UM001 and UM004 cells were treated with increasing doses of LY2801653 and LY2875358 followed by HGF stimulation. Both cMET inhibitors effectively blocked HGF-induced phosphorylation of ERK1/2, AKT, and cMET at tyrosine 1349 (Fig. 3A and 3B). Phosphorylation at tyrosine 1349 in the cMET cytoplasmic domain provides a direct binding site for Gab1 (36), which promotes AKT pathway activation. Of note, LY2875358 had minimal effect on HGF-induced phosphorylation of cMET at tyrosine 1234/5 (Fig. 3A and B), critical sites for kinase activation. We evaluated the ability of these 2 cMET targeting agents in overcoming HGF-mediated resistance to trametinib in uveal melanoma cells. LY2801653 alone did not significantly alter UM001 and UM004 cell growth at 100 nmol/L; however, growth of trametinib-treated uveal melanoma cells which decreased by about 57% compared with the vehicle control was further inhibited when treated with LY2801653 (Fig. 3B). The viability of trametinib/LY2801653 cotreated UM001 and UM004 cells decreased by 81% and 64%, respectively, of the vehicle control. Importantly, HGF-mediated growth protection from trametinib treatment was abrogated by LY2801653 (Fig. 3B). Similarly, LY2875358 alone had little effects on UM001 and UM004 cell growth. Although LY2875358 did not further inhibit growth of trametinib-treated cells, LY2875358 blocked HGF-mediated protection from trametinib (Fig. 3C). The viability of trametinib-treated UM001 and UM004 cells was increased to levels similar to the vehicle control when cells were treated with HGF; an effect that was decreased with LY2875358 by 44% in UM001 and 25% in UM004 compared with vehicle control (Fig. 3C). Together, these data demonstrate that targeting HGF signaling with clinical-grade cMET neutralizing antibody and inhibitor overcomes HGF-mediated resistance to trametinib in metastatic uveal melanoma cells.

Basal phosphorylation of cMET and downstream signaling is low in uveal melanoma lines, and understanding the communication between cancer cells and the stroma in the metastatic site is necessary for the development of optimal therapeutic regimens. Uveal melanoma frequently metastasizes to the liver. Hepatic stellate cells are intralobular connective tissue cells that are quiescent in a healthy liver but transition into myofibroblast-like cells and become activated during liver fibrosis and hepatocellular carcinomas (37). Current available stellate cell lines are either immortalized by hTERT or become activated because of long-time culture and therefore at least partially lose characteristics of their primary origins (38). Therefore, we utilized primary stellate cells that were isolated from human liver. These cells were cultured for up to 6 passages to minimize their activation, passages at which they did not express the fibroblast markers, α-SMA and FAP (Supplementary Fig. S3A).

To better understand the effects of hepatic stellate cells on uveal melanoma cells, we first performed high-throughput antibody-based RPPA analysis on UM001 and UM004 cells incubated for 1 or 48 hours with either unconditioned medium or stellate cell conditioned medium. Supervised clustering of proteins that were regulated by stellate cell conditioned medium and further significance analysis of microarrays (SAM) identified several proteins that were differentially regulated by addition of stellate cell conditioned medium (Fig. 4A; Supplementary Fig. S3B). In both UM001 and UM004 cells, PI3K/AKT and ERK/MAPK signaling were the most activated pathways by stellate cell medium (Fig. 4A). We performed Western blot analysis to validate the RPPA findings. In UM001 and UM004 cells, conditioned medium from stellate cells rapidly induced phosphorylation of ERK1/2, AKT, cMET, and Stat3 (Fig. 4B). We also demonstrated that HGF was present at ng/mL levels in the conditioned medium from early passages of primary hepatic stellate cells by ELISA (Supplementary Fig. S3C). In contrast, the conditioned medium from an immortalized human hepatic stellate cell line did not induce cMET activation (Supplementary Fig. S3D). Consistent with the known role of HGF (39), we showed that stellate cell conditioned medium promoted the migration and invasion of UM001 and UM004 cells (Supplementary Fig. S4).

To determine whether conditioned medium from stellate cells drives resistance to trametinib though HGF/cMET pathway activation, we cultured UM001 and UM004 cells in either unconditioned medium or stellate cell conditioned medium. Factors from stellate cells protected uveal melanoma cells from trametinib-induced growth inhibition, as the viability of uveal melanoma cells cultured in conditioned medium increased by 2- to 3-fold compared with trametinib treatment/nonconditioned medium conditions (Fig. 4C). Importantly, stellate cell conditioned medium protection to trametinib was restored by LY2875358 and LY2801653, with LY2801653 being more potent in sensitizing uveal melanoma cells (85%–90% reduction in cell viability compared with the vehicle control). This suggests that LY2801653 is a more effective cMET inhibitor, and/or signaling molecules other than cMET may play a role in response to trametinib in uveal melanoma cells (Fig. 4C). However, together these data indicate that factors from hepatic stellate cells elicit innate resistance to trametinib at least partially through HGF/cMET signaling.

To test whether combined therapies targeting MEK1/2 and HGF/cMET signaling improve the response in metastatic uveal melanoma, we next extended our study to analyze a mutant GNAQ harboring uveal melanoma patient sample using an ex vivo treatment approach (Fig. 6A; Supplementary Table S1). Tumor tissue pieces were treated with DMSO, trametinib, LY2875358, or a combination of trametinib and LY2875358. As expected, treatment with trametinib inhibited the phosphorylation of ERK1/2 (Fig. 6B), suggesting that ex vivo treatment of patient-derived explants is a feasible strategy for testing drug response in uveal melanoma. Ex vivo treatment with trametinib also promoted apoptosis as evidenced by an increased expression of cleaved PARP. Interestingly, combination of trametinib with LY2875358 further upregulated the expression of cleaved PARP. These data are supportive that a combined therapy with MEK and cMET inhibition may represent a novel and effective strategy in treating patients with metastatic uveal melanoma.

Discussion

The majority of uveal melanoma metastases show a tropism for the liver and are highly resistant to targeted therapies such as MEK inhibitors. How the tumor microenvironment regulates the response in uveal melanoma to targeted inhibitors is poorly understood. Here, we utilized cell lines derived from metastatic uveal melanoma and conditioned medium derived from stromal cells in the liver microenvironment. We provide evidence that the use of cMET targeting agents as a part of combinational approach may counteract tumor microenvironment-mediated primary resistance to MEK inhibitors in mutant GNAQ/11 metastatic uveal melanoma.

Recent results from the phase III, randomized trial (NCT01974752) of the MEK inhibitor, selumetinib, in combination with dacarbazine in patients with metastatic uveal melanoma were disappointing with only 3 of 97 patients treated with the combination eliciting a partial response on the basis of a central review. These results are in contrast to findings in cutaneous melanoma, which led to the FDA approval of trametinib for the treatment of BRAF V600E/K unresectable or metastatic cutaneous melanoma (40). HGF is abundant in the liver microenvironment and, when supplied exogenously, rescues the growth of MEK-inhibited mutant GNAQ human metastatic uveal melanoma cell lines (20). Our data herein indicate that HGF-mediated resistance to MEK inhibitors in uveal melanoma cells involves silencing of the proapoptotic Bim-EL and Bmf. These data are similar to the role of Bim-EL and Bmf in resistance to the BRAF inhibitor, PLX4720, in cutaneous melanoma cells (41).

To investigate the effect of liver microenvironment on response to MEK inhibitors in uveal melanoma cells, we examined factors derived from human hepatic stellate cells. Early-passage human stellate cells do not display activation markers and do secrete HGF, indicating that they may be an appropriate model for studying stromal contributions from the metastatic uveal melanoma tumor microenvironment. Pro-HGF is subsequently cleaved to form HGF, which acts as a growth factor for hepatocytes (42). cMET is expressed in both primary and metastatic uveal melanomas, but metastatic lesions tend to have higher cMET expression levels (43), which is activated in the majority of uveal melanoma liver metastases (20). Indeed, cMET signaling is constitutively activated in uveal melanoma cells when cultured in conditioned medium from stellate cells. These data support a role for tumor microenvironment in regulating HGF/cMET signaling in metastatic uveal melanoma, which is mediated by stellate cell–cancer cell communication in the liver.

While uveal melanoma cells are sensitive to trametinib in regular growth medium, they are resistant when grown in conditioned medium derived from stellate cells. Importantly, resistance is overcome by cMET targeting agents. These data suggest that innate/intrinsic resistance of uveal melanoma to MEK inhibitors is driven, at least in part, by HGF from stellate cells in the liver microenvironment. We demonstrate that cMET targeting agents such as LY2801653 and LY2875358 may improve the response to MEK inhibitors in metastatic patients with uveal melanoma. We extended our studies to analyze a uveal melanoma surgical specimen in an ex vivo treatment approach, which maintains the tumor microenvironment. Interestingly, we observed that LY2801653 treatment promoted the expression of cleaved PARP, an indicator of apoptosis. We acknowledge that further studies using preclinical models are important to address the effect of combinational MEK1/2 and cMET–based target therapy in metastatic uveal melanoma.

The main activated downstream pathway of HGF/cMET is PI3K/AKT signaling. In the presence of trametinib, HGF promotes the activation of PI3K/AKT, which compensates for loss of MEK–ERK1/2 activity in uveal melanoma cells (20). Despite the evidence highlighting the importance of the PI3K pathway activation in the development of resistance to targeted therapy in melanoma, initial testing of class I PI3K inhibitors in patients has not produced dramatic results mainly due to the overlapping toxicities with MEK inhibitors that limits their effective dosing (44). One possible way to overcome this limitation is to utilize PI3K isoform–specific inhibitors. We identified that PI3Kα/γ/δ isoforms, but not PI3Kβ, are responsible for HGF-mediated AKT activation and HGF-mediated resistance to MEK inhibitors. These data suggest that the use of PI3Kβ-sparing inhibitors may represent a useful strategy to overcome HGF-mediated resistance and subsequently improve responses to MEK inhibitors in metastatic uveal melanoma. Of note, in cutaneous BRAF-mutated GEM melanoma models, the combination of MEK inhibitor plus the PI3Kβ-sparing inhibitor enhanced initial tumor regression and forestalled the onset of tumor resistance (45).

In summary, the data presented here show for the first time that stellate cells from the liver provide innate resistance to MEK inhibitors in metastatic uveal melanoma through HGF/cMET signaling. We have provided evidence that downregulation of the BH3-only proteins, Bim-EL and Bmf, contributes to HGF-mediated resistance. Blocking HGF signaling with either clinical-grade cMET targeting agents or PI3Kα/γ/δ inhibitors in uveal melanoma cells overcome resistance to MEK inhibitors mediated by stellate cells or exogenous HGF. Ongoing efforts include testing anti-cMET monoclonal antibodies in combination with MEK inhibitors in preclinical uveal melanoma studies. In addition, profiling other factors within the hepatic cellular architecture that regulate response to targeted therapy may identify novel targets for more effective therapeutic strategies.

Disclosure of Potential Conflicts of Interest

M.A. Davies reports receiving commercial research grants from GlaxoSmithKline, AstraZeneca, Roche/Genentech, and Sanofi-Aventis and is a consultant/advisory board member for Novartis, Roche/Genentech, GlaxoSmithKline, Sanofi-Aventis, and Vaccinex. T. Sato reports receiving commercial research grant from Eli Lilly and Company and other commercial research support from Guerbet LLC and is a consultant/advisory board member for Immunocore. A.E. Aplin reports receiving commercial research grant from Pfizer. No potential conflicts of interest were disclosed by the other authors.

Grant Support

This project was funded by a Dr. Ralph and Marian Falk Medical Research Trust Catalyst Award, a Programmatic Initiative Award from the Provost's office at Thomas Jefferson University, and CURE award funding from Pennsylvania Department of Health. The Sidney Kimmel Cancer Center Flow Cytometry and Genomics core facilities are supported by NIH/National Cancer Institute Support Grant (P30 CA056036). The RPPA studies were supported by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation and were performed at the Functional Proteomics Core Facility at MD Anderson Cancer Center, which is supported by a National Cancer Institute (NCI) Cancer Center Support Grant (CA-16672). Dr. Andrew E. Aplin is also supported by NIH R01 (CA-182635) and a grant from the Pennsylvania Department of Health. The CCBS program is supported by the Cancer Center Support Grant (5P30CA056036-17).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Acknowledgments

LY2801653 and LY2875358 were generously provided by Eli Lilly and Company.

Footnotes

Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

LY2801653 is an orally bioavailable multi-kinase inhibitor with potent activity against MET, MST1R, and other oncoproteins, and displays anti-tumor activities in mouse xenograft models.
Invest New Drugs 2013;31:833–44.